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The aim of this research was to evaluate the influence of maternal cells from colostrum on the development and function of the innate immune response in Holstein calves. Calves were divided into 2 groups: COL+ (n=10) received fresh colostrum; and COL- (n=10) which received frozen colostrum containing no viable cells. The calves were assessed before colostrum intake (D0), 48h of age (D2), and weekly from D7 up to D28. Blood samples were collected for analysis of the distribution of leukocytes, cellular phenotype and in vitro granulocyte function. COL+ calves tended to have a high number of neutrophils on D7 (p=0.073). COL- calves took up significantly more Escherichia coli (measured as MFI) on D7 (p=0.034). Endogenous production of radicals (as percentage of cells) tended to be higher in COL- calves on D14 (p=0.061). The intensity of endogenous reactive oxygen species (ROS) produced by granulocytes tended to be higher in COL+ calves on D21 (p=0.094). Overall, ROS production (percent of cells, and MFI) induced by Staphylococcus aureus and Escherichia coli were higher in COL+ calves than COL- calves. It was our observation that COL+ calves developed an innate immune response more quickly and efficiently after natural exposure to pathogens after birth. In contrast, COL- calves mounted an innate response more slowly that yielded a persistent inflammatory response after natural exposure to these bacteria agents. This research provides evidence of an advantage to the calf of receiving fresh colostrum on the development and function of the innate immune system., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Platelets are released from megakaryocytes. The bone marrow has been proposed to be the major site where this process occurs. Lefrançais et al. (2017) using state-of-the-art techniques including two-photon microscopy, in vivo lineage-tracing technologies, and sophisticated lung transplants reveal that the lung is also a primary site for platelet biogenesis. Strikingly, lung megakaryocytes can completely reconstitute platelet counts in the blood in mice with thrombocytopenia. This study also shows that hematopoietic progenitors, with capacity to repopulate the bone marrow after irradiation, are present in the lungs. This work brings a novel unexpected role for the lung as a niche for hematopoiesis. The emerging knowledge from this research may be important for the treatment of several disorders.

Objectives: Pituitary-dependent hyperadrenocorticism is the most common cause of naturally occurring hypercortisolism in dogs. CRHR1 expression in human and dog corticotrophinomas suggested that this gene affects pituitary tumorigenesis. The present study aimed to investigate mutations in the CRHR1 coding region in poodles with pituitary-dependent hyperadrenocorticism., Methods: Fifty poodles with pituitary-dependent hyperadrenocorticism and 50 healthy poodles were studied. Genomic DNA was amplified by PCR and analyzed by Sanger sequencing., Results: The novel CRHR1 p.V97M mutation was identified in one dog. This valine residue, located in the amino-terminal extracellular domain, exhibits high affinity for its corticotropin-releasing hormone (CRH) ligand. Bioinformatic analysis revealed structural rearrangements in the mutant protein, with a 17% increase in the surface binding affinity between CRHR1 and CRH. In vitro functional studies showed that mutant CRHR1 induced higher ACTH secretion than the wild type after stimulation with human CRH., Conclusion: These results suggest that germline activating mutations in CRHR1 may be a rare cause of pituitary hyperadrenocorticism in poodles.

Improved performances in gynaecological ultrasonography have enabled an increasing number of often asymptomatic endometrial polyps to be detected. Most of these polyps are removed surgically, as a precautionary measure, so as not to miss a case of endometrial cancer. Nonetheless, this management strategy is based solely on the sonographer's judgement and a number of these operations, which are probably of no benefit, could be avoided. In order to do so, risk factors for malignancy need to be identified., Objective: Estimate the prevalence of lesions in menopausal patients with a pre-operative diagnosis of endometrial polyp. Establish risk factors for malignancy., Study Design: This is a single-centre retrospective study. Enrolment criteria were menopausal patients aged over 45 who had undergone hysteroscopic resection of a polyp. Pre-op diagnosis was made either by ultrasonography or diagnostic hysteroscopy. Malignant lesions included cancers and atypical hyperplasia. Benign lesions consisted of simple polyps, non-atypical simple hyperplasia and non-atypical complex hyperplasia. Risk factors studied were existing abnormal uterine bleeding, endometrial thickness, personal or first-degree family history of gynaecological cancer (breast, cervix, endometrium, ovary) and age on diagnosis., Results: 631 patients were enrolled of whom 30 presented a malignant disorder (4.75%); 579 patients (91.76%) presented a simple polyp, 11 a non-atypical simple hyperplasia (1.74%) and 11 a non-atypical complex hyperplasia (1.74%). On univariate analysis age alone proved to be statistically significant (OR 1.05; 95%CI=[1.02-1.09] p<0.01), with a threshold of 59 years of age on the ROC curve. On multivariate analysis, factors predictive of a malignant lesion were age (OR=1.06; 95%CI [1.02-1.10]), existence of AUB (OR=2.4; 95% CI [1.07-5.42]) and family history (OR=2.88; 95%CI [1.08-7.67]). Neither the univariate nor multivariate model was able to demonstrate a statistically significant relationship with respect to endometrial thickness. The risk of malignancy was 12.3% in patients aged over 59 presenting AUB. For all other subgroups, the risk varied between 2.31 and 3.78%., Conclusion: The risk of a malignant lesion appears to be high (12%) in menopausal patients aged over 59 presenting an endometrial polyp detected when there is pre-existing AUB. In this situation, hysteroscopic resection of endometrial polyps should therefore be routinely proposed. For other patients, the risk of a malignant lesion is low but not insignificant, standing at about 3%. Each patient record should therefore be discussed on an individual case basis, taking into consideration the patient's pre-existing conditions, after providing clear and appropriate information., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP., Methods: Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP., Results: In this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors  to validate this new luminescence method., Conclusion: With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.

The objective of this research was to evaluate the influence of cells from colostrum on the health of neonate calves. Animals were distributed in 2 groups: COL+ (n=9) which received fresh colostrum from their own damns; and COL- (n=10) which received frozen colostrums from donors. Heifers were assessed before colostrum intake - D0; D2; D7; D14; D21 and D28. Heifers were monitored by clinical examination, hematological profile and serum iron. COL- had a higher diarrhea intensity score (typically 3) on D7. Moreover, a single case each of bronchopneumonia and navel inflammation were observed in COL- calves. COL- had fewer red blood cells (RBC) (6.5±0.8×10 6 /μL) and less hemoglobin (Hgb) (8.3±1.4g/dL) than COL+ (RBC=7.2±0.8×10 6 /μL; Hgb=9.6±1.3g/dL) at D14 (P≤0.05). COL- had more anemia on D21 (P=0.03) and on D28 (P=0.02). Iron was lower in COL- (5.6±2.7μM/L) than COL+ (10.7±6.2μM/L) (P=0.03) on D7. Lymphocytes was lower in COL- than COL+ on D7 (3.8±1.0×10 3 /μL COL+ and 5.4±2.2×10 3 /μL COL-, P=0.02). COL- calves had more anemia and lower serum iron concomitant with diarrhea on D7. The number of leukocytes was relatively consistent in the COL+ calves, while COL- calves showed an increasing number of of lymphocytes starting on D7., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The clinical benefits of short-term therapy with glucocorticoids (GC) in patients with inflammatory bowel disease (IBD) are widely known. However, the effects of this treatment towards the re-establishment of the regulatory network in IBD are not fully explored. We have evaluated the immunological effects of the abbreviated GC therapy in experimental colitis induced by 3% dextran sulphate sodium in C57BL/6 mice. Treatment with GC improved disease outcome, constrained circulating leucocytes and ameliorated intestinal inflammation. The control of the local inflammatory responses involved a reduction in the expression of interferon-γ and interleukin-1β, associated with augmented mRNA levels of peroxisome proliferator-activated receptors (α and γ) in intestine. Furthermore, there was a reduction of CD4 + T cells producing interferon-γ, together with an increased frequency of the putative regulatory population of T cells producing interleukin-10, in spleen. These systemic alterations were accompanied by a decrease in the proliferative potential of splenocytes of mice treated in vivo with GC. Notably, treatment with GC also led to an increase in the frequency of the regulatory markers GITR, CTLA-4, PD-1, CD73 and FoxP3, more prominently in spleen. Taken together, our results pointed to a role of GC in the control of leucocyte responsiveness and re-establishment of a regulatory system, which probably contributed to disease control and the restoration of immune balance. Finally, this is the first time that GC treatment was associated with the modulation of a broad number of regulatory markers in an experimental model of colitis., (© 2016 John Wiley & Sons Ltd.)

Dehydroepiandrosterone (DHEA) is a hormone that plays an important role in the modulation of inflammatory responses. However, the precise mechanisms that link the actions of this androgen with protection or susceptibility to inflammatory bowel diseases (IBD) remain uknown. Here we showed that low dose DHEA inhibited proliferation of spleen cells and IFN-у production. The hormone was not toxic to myeloid lineage cells, although it caused necrosis of spleen cells at the intermediate and highest doses in vitro (50 and 100μM). The treatment of C57BL/6 mice with DHEA during colitis induction by dextran sodium sulfate (DSS) led to a reduction in weight loss and clinical signs of disease. There were decreased peripheral blood monocytes on day 6 of DSS exposure and treatment, besides increase in circulating neutrophils in the tissue repair phase. DHEA also led to reduced lamina propria cellularity and restoration of normal colon length. These results were accompanied by decreased expression of IL-6 and TGF-β mRNA, while IL-13 was augmented in the colon on day 6, which was probably related to attenuation of inflammation. There was retention of CD4(+) cells in the spleen after use of DHEA, along with augmented frequency of CD4(+)IL-4(+) cells, decreased CD4(+)IFN-ɣ(+) in spleen and constrained CD4(+)IL-17(+) population in the mesenteric lymph nodes. Moreover, splenocytes of mice treated with DHEA became hyporesponsive, as observed by reduced proliferation after re-stimulation ex-vivo. In conclusion, DHEA modifyies leukocyte activity and balances the exacerbated immune responses which drive local and systemic damages in IBD., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Myocardial iron quantification remains limited to 1.5 T systems with T2* measurement. The present study aimed at comparing myocardial T2* values at 1.5 T to T1 and T2 mapping at 3.0 T in patients with iron overload and healthy controls. A total of 17 normal volunteers and seven patients with a history of myocardial iron overload were prospectively enrolled. Mid-interventricular septum T2*, native T1 and T2 times were quantified on the same day, using a multi-echo gradient-echo sequence at 1.5 T and T1 and T2 mapping sequences at 3.0 T, respectively. Subjects with myocardial iron overload (T2* < 20 ms) in comparison with those without had significantly lower mean myocardial T1 times (868.9 ± 120.2 vs. 1170.3 ± 25.0 ms P = 0.005 respectively) and T2 times (34.9 ± 4.7 vs. 45.1 ± 2.0 ms P = 0.007 respectively). 3 T T1 and T2 times strongly correlated with 1.5 T, T2* times (Pearson's r = 0.95 and 0.91 respectively). T1 and T2 measures presented less variability than T2* in inter- and intra-observer analysis. Native myocardial T1 and T2 times at 3 T correlate closely with T2* times at 1.5 T and may be useful for myocardial iron overload quantification.

The adrenal glands are able to modulate immune responses through neuroimmunoendocrine interactions and cortisol secretion that could suppress exacerbated inflammation such as in inflammatory bowel disease (IBD). Therefore, here we evaluated the role of these glands in experimental colitis induced by 3% dextran sulfate sodium (DSS) in C57BL/6 mice subjected to adrenalectomy, with or without glucocorticoid (GC) replacement. Mice succumbed to colitis without adrenals with a higher clinical score and augmented systemic levels of IL-6 and lower LPS. Furthermore, adrenalectomy negatively modulated systemic regulatory markers. The absence of adrenals resulted in augmented tolerogenic lamina propria dendritic cells but no compensatory local production of corticosterone and decreased mucosal inflammation associated with increased IFN-γ and FasL in the intestine. To clarify the importance of GC in this scenario, GC replacement in adrenalectomized mice restored different markers to the same degree of that observed in DSS group. Finally, this is the first time that adrenal-derived hormones, especially GC, were associated with the differential local modulation of the gut infiltrate, also pointing to a relationship between adrenalectomy and the modulation of systemic regulatory markers. These findings may elucidate some neuroimmunoendocrine mechanisms that dictate colitis outcome.

Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue-green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera., (Copyright © 2015 John Wiley & Sons, Ltd.)

The aim of this study was to evaluate the potential of the successive projection algorithm for interval selection in partial least squares (iSPA-PLS) together with near-infrared reflectance spectroscopy (NIRS) as a feasible method to determine the total anthocyanin content (TAC) of intact jaboticaba fruit [Myrciaria jaboticaba (Vell.) O. Berg]. A total of 579 jaboticaba fruit were collected in three different harvests in three separate years (2011 and 2013). The correlation coefficients between the predicted and measured TAC were between 0.65 and 0.89, the RMSEPs were 7.55 g kg(-1) and 9.35 g kg(-1) (good accuracy) for prediction set, respectively. The RPD ratios for TAC were in the range of 2.57-3.19 with iSPA-PLS, which showed better predictive performance (acceptable precision). These results suggest that the NIR spectroscopy and wavelength selection (iSPA-PLS) algorithm can be used to determine the TAC of intact jaboticaba fruit., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Current therapies for inflammatory bowel disease (IBD) are not totally effective, resulting in persistent and recurrent disease for many patients. Mosquito saliva contains immunomodulatory molecules and therein could represent a novel therapy for IBD. Here, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Aedes aegypti on dextran sulfate sodium (DSS)-induced colitis. For this purpose, C57BL/6 male mice were exposed to 3% DSS in drinking water and treated with SGE at early (days 3-5) or late (days 5-8) time points, followed by euthanasia on days 6 and 9, respectively, for sample collection. The results showed an improvement in clinical disease outcome and postmortem scores after SGE treatment, accompanied by the systemic reduction in peripheral blood lymphocytes, with no impact on bone marrow and mesenteric lymph nodes cellularity or macrophages toxicity. Moreover, a local diminishment of IFN-γ, TNF-α, IL-1β and IL-5 cytokines together with a reduction in the inflammatory area were observed in the colon of SGE-treated mice. Strikingly, early treatment with SGE led to mice protection from a late DSS re-challenging, as observed by decreased clinical and postmortem scores, besides reduced circulating lymphocytes, indicating that the mosquito saliva may present components able to prevent disease relapse. Indeed, high performance liquid chromatography (HPLC) experiments pointed to a major SGE pool fraction (F3) able to ameliorate disease signs. In conclusion, SGE and its components might represent a source of important immunomodulatory molecules with promising therapeutic activity for IBD., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi-only 71 species, all within the ∼ 9,000 fungi of the temperate and tropical Agaricales order-are reported from among ∼ 100,000 described fungal species [6, 7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a byproduct of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature-compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millennia [8-15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin "mushrooms," internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy, where wind flow is greatly reduced., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The Malpighian tubules play a key role in insect osmoregulation. Although a transcriptional analysis has been done for the Malpighian tubules in Drosophila melanogaster (Diptera), no functional genomics analysis has yet been carried out for any Coleoptera species. Recently, we constructed a cDNA library from Malpighian tubules of larval Zophobas morio, a close relative of Tribolium castaneum, and cloned the cDNA for an AMP/CoA-ligase with luciferase-like enzyme properties. Using this cDNA library, we randomly isolated, partially sequenced and analyzed ca. 540 clones, obtaining the first transcriptional profile of the most representative expressed genes, and associated them with their possible biological functions. A high percentage of mitochondrial genes was found, which is consistent with the high metabolic activity required by this organ during the formation of primary urine. Common transcripts included those for enzymes involved in osmoregulation, such as solute transporters and ATPases, and in detoxification and excretion, such as cytochrome P450, glutathione S-transferase, alcohol dehydrogenase. The presence of AMP/CoA-ligases, which activate exogenous carboxylic acids such as firefly D-luciferin suggests their participation in important new xenobiotic excretion/detoxification roles in Malpighian tubule physiology.

Various studies have indicated that music therapy with relaxant music improves cardiac function of patients treated with cardiotoxic medication and heavy-metal music acutely reduces heart rate variability (HRV). There is also evidence that white noise auditory stimulation above 50 dB causes cardiac autonomic responses. In this study, we aimed to evaluate the acute effects of musical auditory stimulation with different intensities on cardiac autonomic regulation. This study was performed on 24 healthy women between 18 and 25 years of age. We analyzed HRV in the time [standard deviation of normal-to-normal RR intervals (SDNN), percentage of adjacent RR intervals with a difference of duration >50 ms (pNN50), and root-mean square of differences between adjacent normal RR intervals in a time interval (RMSSD)] and frequency [low frequency (LF), high frequency (HF), and LF/HF ratio] domains. HRV was recorded at rest for 10 minutes. Subsequently, the volunteers were exposed to baroque or heavy-metal music for 5 minutes through an earphone. The volunteers were exposed to three equivalent sound levels (60-70, 70-80, and 80-90 dB). After the first baroque or heavy-metal music, they remained at rest for 5 minutes and then they were exposed to the other music. The sequence of songs was randomized for each individual. Heavy-metal musical auditory stimulation at 80-90 dB reduced the SDNN index compared with control (44.39 ± 14.40 ms vs. 34.88 ± 8.69 ms), and stimulation at 60-70 dB decreased the LF (ms(2)) index compared with control (668.83 ± 648.74 ms(2) vs. 392.5 ± 179.94 ms(2)). Baroque music at 60-70 dB reduced the LF (ms(2)) index (587.75 ± 318.44 ms(2) vs. 376.21 ± 178.85 ms(2)). In conclusion, heavy-metal and baroque musical auditory stimulation at lower intensities acutely reduced global modulation of the heart and only heavy-metal music reduced HRV at higher intensities.

The aim of this study was to evaluate the potential of near-infrared reflectance spectroscopy (NIR) as a rapid and non-destructive method to determine soluble solid content (SSC) in intact jaboticaba [Myrciaria jaboticaba (Vell.) O. Berg] fruit. Multivariate calibration techniques were compared with pre-processed data and variable selection algorithms, such as partial least squares (PLS), interval partial least squares (iPLS), a genetic algorithm (GA), a successive projections algorithm (SPA) and nonlinear techniques (BP-ANN, back propagation of artificial neural networks; LS-SVM, least squares support vector machine) were applied to building the calibration models. The PLS model produced prediction accuracy (R(2)=0.71, RMSEP=1.33 °Brix, and RPD=1.65) while the BP-ANN model (R(2)=0.68, RMSEM=1.20 °Brix, and RPD=1.83) and LS-SVM models achieved lower performance metrics (R(2)=0.44, RMSEP=1.89 °Brix, and RPD=1.16). This study was the first attempt to use NIR spectroscopy as a non-destructive method to determine SSC jaboticaba fruit., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The development of sensitive methodologies for detecting agrochemicals has become important in recent years due to the increasingly indiscriminate use of these substances. In this context, nanosensors based on atomic force microscopy (AFM) tips are useful because they provide higher sensitivity with operation at the nanometer scale. In this paper we exploit specific interactions between AFM tips functionalized with the enzyme acetolactate synthase (ALS) to detect the ALS-inhibitor herbicides metsulfuron-methyl and imazaquin. Using atomic force spectroscopy (AFS) we could measure the adhesion force between tip and substrate, which was considerably higher when the ALS-functionalized tip (nanobiosensor) was employed. The increase was approximately 250% and 160% for metsulfuron-methyl and imazaquin, respectively, in comparison to unfunctionalized probes. We estimated the specific enzyme-herbicide force by assuming that the measured force comprises an adhesion force according to the Johnson-Kendall-Roberts (JKR) model, the capillary force and the specific force. We show that the specific, biorecognition force plays a crucial role in the higher sensitivity of the nanobiosensor, thus opening the way for the design of similarly engineered tips for detecting herbicides and other analytes.

Background: No clear evidence is available in the literature regarding the acute effect of different styles of music on cardiac autonomic control., Objectives: The present study aimed to evaluate the acute effects of classical baroque and heavy metal musical auditory stimulation on Heart Rate Variability (HRV) in healthy men., Patients and Methods: In this study, HRV was analyzed regarding time (SDNN, RMSSD, NN50, and pNN50) and frequency domain (LF, HF, and LF / HF) in 12 healthy men. HRV was recorded at seated rest for 10 minutes. Subsequently, the participants were exposed to classical baroque or heavy metal music for five minutes through an earphone at seated rest. After exposure to the first song, they remained at rest for five minutes and they were again exposed to classical baroque or heavy metal music. The music sequence was random for each individual. Standard statistical methods were used for calculation of means and standard deviations. Besides, ANOVA and Friedman test were used for parametric and non-parametric distributions, respectively., Results: While listening to heavy metal music, SDNN was reduced compared to the baseline (P = 0.023). In addition, the LF index (ms(2) and nu) was reduced during exposure to both heavy metal and classical baroque musical auditory stimulation compared to the control condition (P = 0.010 and P = 0.048, respectively). However, the HF index (ms(2)) was reduced only during auditory stimulation with music heavy metal (P = 0.01). The LF/HF ratio on the other hand decreased during auditory stimulation with classical baroque music (P = 0.019)., Conclusions: Acute auditory stimulation with the selected heavy metal musical auditory stimulation decreased the sympathetic and parasympathetic modulation on the heart, while exposure to a selected classical baroque music reduced sympathetic regulation on the heart.

We investigated the acute effects of musical auditory stimulation on cardiac autonomic responses to a mental task in 28 healthy men (18-22 years old). In the control protocol (no music), the volunteers remained at seated rest for 10 min and the test was applied for five minutes. After the end of test the subjects remained seated for five more minutes. In the music protocol, the volunteers remained at seated rest for 10 min, then were exposed to music for 10 min; the test was then applied over five minutes, and the subjects remained seated for five more minutes after the test. In the control and music protocols the time domain and frequency domain indices of heart rate variability remained unchanged before, during and after the test. We found that musical auditory stimulation with baroque music did not influence cardiac autonomic responses to the mental task., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae., (Copyright © 2013 John Wiley & Sons, Ltd.)

The açaí (Euterpe oleracea Mart.) and juçara (Euterpe edulis Mart.) produce similar fruits which are rich in energy, minerals, vitamins and natural compounds with antioxidant and anti-inflammatory properties. Although the drink obtained from these species is similar, it is important to develop tools to establish the identity of the fruit species and growing regions. To assess claims of origin and for other purposes, we use multivariate analysis to investigate the differentiation of açaí and juçara fruits based on rare earth element (REE) content determined by Inductively Coupled Plasma Mass Spectrometry. REE content, in particular Sm, Th, La, Pr, Gd, and especially Ce and Nd varied between species. PCA analysis was not efficient in differentiating açaí from juçara fruit samples. In contrast, LDA analysis permitted a correct differentiation between species with a predictive ability of 83.3%. The methodology that we have applied confirms that REE can be used to differentiate between açaí and juçara fruit samples and to identify their origin., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Manganese (Mn) is an essential element for human health. However, at high concentrations Mn may be neurotoxic. Mn accumulates in astrocytes, affecting their redox status. In view of the high antioxidant and anti-inflammatory properties of the exotic Brazilian fruit açaí (Euterpe oleracea Mart.), its methanolic extract was obtained by solid-phase extraction (SPE). This açaí extract showed considerable anthocyanins content and direct antioxidant capacity. The açaí extract scavenged 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH•) with an EC₅₀ of 19.1 ppm, showing higher antioxidant activity compared to butylated hydroxytoluene (BHT), but lower than ascorbic acid and quercetin. This obtained açaí extract also attenuated Mn-induced oxidative stress in primary cultured astrocytes. Specifically, the açaí extract at an optimal and nutritionally relevant concentration of 0.1 μg/ml prevented Mn-induced oxidative stress by (1) restoring GSH/GSSG ratio and net glutamate uptake, (2) protecting astrocytic membranes from lipid peroxidation, and (3) decreasing Mn-induced expression of erythroid 2-related factor (Nrf2) protein. A larger quantity of açaí extract exacerbated the effects of Mn on these parameters except with respect to lipid peroxidation assessed by means of F₂-isoprostanes. These studies indicate that at nutritionally relevant concentration, anthocyanins obtained from açaí protect astrocytes against Mn neurotoxicity, but at high concentrations, the "pro-oxidant" effects of its constituents likely prevail. Future studies may be profitably directed at potential protective effects of açaí anthocyanins in nutraceutical formulations.

Background: Patients with Sickle cell disease (SCD) who receive regular transfusions are at risk for developing cardiac toxicity from iron overload. The aim of this study was to assess right and left cardiac volumes and function, late gadolinium enhancement (LGE) and iron deposits in patients with SCD using CMR, correlating these values with transfusion burden, ferritin and hemoglobin levels., Methods: Thirty patients with SCD older than 20 years of age were studied in a 1.5 T scanner and compared to age- and sex-matched normal controls. Patients underwent analysis of biventricular volumes and function, LGE and T2* assessment of the liver and heart., Results: When compared to controls, patients with SCD presented higher left ventricular (LV) volumes with decreased ejection fraction (EF) with an increase in stroke volume (SV) and LV hypertrophy. The right ventricle (RV) also presented with a decreased EF and hypertrophy, with an increased end-systolic volume. Although twenty-six patients had increased liver iron concentrations (median liver iron concentration value was 11.83 ± 9.66 mg/g), only one patient demonstrated an abnormal heart T2* < 20 msec. Only four patients (13%) LGE, with only one patient with an ischemic pattern., Conclusions: Abnormal heart iron levels and myocardial scars are not a common finding in SCD despite increased liver iron overload. The significantly different ventricular function seen in SCD compared to normal suggests the changes in RV and LV function may not be due to the anemia alone. Future studies are necessary to confirm this association.

Background: The Stroke remains one of the major chronic diseases worldwide, and is considered a major cause of disability, which results not only in persistent neurological deficits, but also in the high physical deconditioning, nevertheless there are not many forms of assessing functional capacity in this population. We aimed to investigate the feasibility of the Six Minute Walk Teste and the Six-Minute Step Test (6MST) in post-stroke patients and compare the behavior of physiological variables during the 6MST and the Six-Minute Walk Test (6MWT), by correlating the functional performance obtained in both tests., Method: The 6MWT was carried out according to the American Thoracic Society (ATS) and the 6MST was performed in six minutes in order to compare it to the 6MWT in a 20 cm step. Was included post-stroke individuals able to walk without aid. All of them did the 6MWT and the 6MST., Results: 12 patients participated in the study. There was no statistical difference in the parameters analyzed when tests were compared. There was poor correlation between the functional performance in both tests., Conclusion: The 6MWT and the 6MST is feasible for post-stroke patients and physiological responses are equal during the performance of both tests. However, there was no correlation with respect to functional performance, which was assessed by the distance walked in the 6MWT and by the number of steps climbed in the 6MST.

Aerophytic cyanobacteria are commonly found growing on rocks, tree trunks and soil, but the diversity of these organisms is still poorly known. This complex group is very problematic considering the taxonomic arrangement and species circumscription, especially when taking into account tropical populations. In this work, 20 samples of aerophytic cyanobacteria from 15 distinct sampling sites were collected along a tropical rainforest area at the São Paulo State (Brazil). Samples were dried at room temperature after the collection, and once in the laboratory, they were rehydrated and analyzed. The taxonomic study resulted in the record and description of nine species of true-branched cyanobacteria pertaining to the genera Spelaeopogon (one specie), Hapalosiphon (two species) and Stigonema (six species). The similarity of the flora found when compared to surveys conducted in other geographical regions was relatively low. These differences could be addressed to ecological conditions of the habitats, to the extension of the area surveyed or even to taxonomic misinterpretations. The molecular assessment of 16S rDNA on the basis of field material was successful for two morphospecies, Hapalosiphon sp. and Stigonema ocellatum; however, their relationships with other populations and species revealed to be uncertain. The results of the floristic survey and of the molecular approach evidenced the fragile delimitation of some genera and species in the true branched group of cyanobacteria.

Introduction: Infants with asymmetric brain lesions are at high risk of developing congenital hemiplegia. Action-observation training (AOT) has been shown to effectively improve upper limb motor function in adults with chronic stroke. AOT is based on action observation, whereby new motor skills can be learnt by observing motor actions. This process is facilitated by the Mirror Neuron System, which matches observed and performed motor actions. This study aims to determine the efficacy of AOT in: (1) influencing the early development of reaching and grasping of typically developing infants and (2) improving the upper limb activity of infants with asymmetric brain lesions., Methods and Analysis: This study design comprises two parallel randomised sham-controlled trials (RCTs) in: (1) typically developing infants (cohort I) and (2) infants with asymmetric brain lesions (eg, arterial stroke, venous infarction, intraventricular haemorrhage or periventricular leukomalacia; cohort II). Cohort II will be identified through a neonatal ultrasound or neonatal MRI. A sham control will be used for both RCTs, taking into consideration that it would be unethical to give no intervention to an at-risk population. Based on a two-tailed t test of two independent means, with a significance (α) level of 0.05, 80% power, predicted effect size of 0.8 and a 90% retention rate, we require 20 participants in each group (total sample of 40) for cohort I. The sample size for cohort II was based on the assumption that the effect size of the proposed training would be similar to that found by Heathcock et al in preterm born infants (n=26) with a mean effect size of 2.4. Given the high effect size, the calculation returned a sample of only four participants per group, on a two-tailed t test, with a significance (α) level of 0.05 and 80% power. As cohort II will consist of two subgroups of lesion type (ie, arterial stroke and venous infarction), we have quadrupled the sample to include 16 participants in each group (total sample of 32). Infants will be randomised to receive either AOT or standard Toy Observation Training (TOT). Both interventions will be of 4 weeks' duration, from the infant's 9th-13th post-term week of age. Three sessions of 5 min each will be performed each day for 6 days/week (total of 6 h over 28 days). Parents of the AOT group will repeatedly show the infant a grasping action on a set of three toys, presented in random order. Parents of the TOT group will show the infant the same set of three toys, in random order, without demonstrating the grasping action. At 14, 16 and 18 weeks, the quantity and quality of reaching and grasping will be measured using the Grasping and Reaching Assessment of Brisbane; symmetry of reaching and grasping will be measured using the Hand Assessment of Infants (HAI) and pressure of grasping for each hand with a customised pressure sensor. At 6 months' corrected age, the primary outcome measures will be the HAI and Bayley Scales of Infant and Toddler Development (third edition; BSID III), to measure cognitive and motor development. At 8 months, HAI and EEG will be used to measure brain activity and cortical coherence. At 12 months, the primary outcome measures will again be HAI and BSID III., Dissemination: This paper outlines the theoretical basis, study hypotheses and outcome measures for two parallel RCTs comparing the novel intervention Action-observation training with standard TOT in: (1) influencing the early development of reaching and grasping of typically developing infants and (2) improving the upper limb motor activity of infants with asymmetric brain lesions., Trial Registration: ACTRN1261100991910. Web address of trial http://www.ANZCTR.org.au/ACTRN12611000991910.aspx.

The use of agrochemicals has increased considerably in recent years, and consequently, there has been increased exposure of ecosystems and human populations to these highly toxic compounds. The study and development of methodologies to detect these substances with greater sensitivity has become extremely relevant. This article describes, for the first time, the use of atomic force spectroscopy (AFS) in the detection of enzyme-inhibiting herbicides. A nanobiosensor based on an atomic force microscopy (AFM) tip functionalised with the acetolactate synthase (ALS) enzyme was developed and characterised. The herbicide metsulfuron-methyl, an ALS inhibitor, was successfully detected through the acquisition of force curves using this biosensor. The adhesion force values were considerably higher when the biosensor was used. An increase of ~250% was achieved relative to the adhesion force using an unfunctionalised AFM tip. This considerable increase was the result of a specific interaction between the enzyme and the herbicide, which was primarily responsible for the efficiency of the nanobiosensor. These results indicate that this methodology is promising for the detection of herbicides, pesticides, and other environmental contaminants.

A series of 3(2H)-furanones, based on side-chain modifications of a parent 3(2H)-furanone, was synthesized in good yield. The parent compound was prepared by hydrogenolysis, and subsequent acid hydrolysis, of isoxazole derivatives. The isoxazole was prepared by a [3+2] 1,3-dipolar cycloaddition reaction between 3-butyn-2-ol and nitrile oxide.

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500 U mL(-1) or 300 Ug(-1) of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme from SSF and designated PL I, II, III, IV, V, and VI, while five peaks were obtained from crude enzyme from SmF and labeled PL I', II', III', IV', and VII'. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35 degrees C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45 degrees C. Crude enzyme from SmF and PL III' showed thermophilic profiles of activity, with maximum activity at 60 and 55 degrees C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0-10.0 and PL III was most stable in the pH range 4.0-7.0. Crude enzyme from SmF retained 70%-80% of its maximum activity in the acid-neutral pH range (4.0-7.0), but PIII showed high stability at alkaline pH (7.5-9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55 degrees C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes.

The Coleoptera order is the richest group among Metazoa, but its phylogenetics remains incompletely understood. Among Coleoptera, bioluminescence is found within the Elateroidea, but the evolution of this character remains a mystery. Mitochondrial DNA has been used extensively to reconstruct phylogenetic relationships, however, the evolution of a single gene does not always correspond to the species evolutionary history and the molecular marker choice is a key step in this type of analysis. To create a solid basis to better understand the evolutionary history of Coleoptera and its bioluminescence, we sequenced and comparatively analyzed the mitochondrial genome of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae).

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases.

Background: Induced sputum (IS) using the cytospin technique has been extensively employed to characterize inflammatory airway diseases; however, procedures of cell enrichment based on cytospin increase the analytical costs and require slide processing within a short period of time after sampling., Study Objectives: To compare three different techniques for cytologic analysis of IS, and to determine the time required by each method and the costs involved., Design: Cross-sectional study., Setting: Tertiary-care university hospital., Patients and Measurements: Eighty-nine patients with asthma and 11 subjects without asthma were submitted to increasing hypertonic saline solution concentrations of 2, 3, 4, and 5% for 7 min for sputum induction. Samples were smeared without treatment with 0.1% dithiothreitol (DTT) [technique A], after treatment with DTT (technique B), and after treatment with DTT and cytospin (technique C). All slides were air-dried and stained with Leishman stain. Two independent observers counted at least 200 inflammatory cells on each slide., Results: Eighty percent of the slides processed by techniques A and B and 65% of the slides processed by technique C represented sputum samples of acceptable quality. The eosinophil percentages in sputum obtained by techniques A and C were closely correlated, as also were those obtained by techniques B and C (r = 0.64 and r = 0.63, respectively; p < 0.01). There was a positive correlation for eosinophils when we compared techniques A and B (r = 0.57, p < 0.01). The neutrophil correlation was significant when the three techniques were compared (technique A vs technique B, r = 0.66; technique A vs technique C, r = 0.51; and technique B vs technique C, r = 0.57; p < 0.01). Bland-Altman analysis showed a good agreement for eosinophil and neutrophil counts when techniques A and B were compared to technique C., Conclusions: The three techniques are good indicators of lung inflammation. Techniques A and B are less time consuming and are of lower cost.

Teller Acuity Cards are a new "preferential looking" procedure for the evaluation of visual acuity in newborns and infants. We used this test to assess, longitudinally, visual acuity in 60 healthy term newborns followed up from birth to two years of age. In order to have a set of comparison parameters for use in studies of newborns at risk of developing visual system impairment, the relative maturational curve was plotted. The acuity values of our sample are in line with those reported by other authors in the literature and they represent the first set of such data referring to a group of healthy term newborns in Italy. This paper provides a visual acuity curve for the first two years of life obtained from healthy term newborns, a curve which could prove useful for reference when this technique is used on newborns at risk of developing neurological and especially visual problems.

The comparison of click beetle and railroadworm luciferases (pH-insensitive) with firefly luciferases (pH-sensitive) showed a set of conserved residues differing between the two groups which could be involved with the bioluminescence spectra pH sensitivity. The substitution C258V in Pyrocoelia miyako (Pml) firefly luciferase and V255C in Ragophthalmus ohbai railroad worm luciferase (Rol) had no effect on the bioluminescence spectra. Substitution of Thr226 in the green-light-emitting luciferases of Rol and Pyrearinus termitilluminans (Pyt) click beetle luciferases resulted in red-shifts (12 to 35 nm), whereas the substitution T226N in the red-light-emitting luciferase of Phrixothrix hirtus (PhRE) railroadworm resulted in a 10 nm blue-shift. In PmL the substitution N230S resulted in a typical red mutant (lambda(max) = 611 nm). The bioluminescence spectrum of all these luciferase mutants did not show altered pH-sensitivity nor considerably changed half-bandwidth in relation to the wild-type luciferases. Altogether present data suggest that Thr226 is an important residue for keeping active-site core in both groups of beetle luciferases. The mechanism for bioluminescence color determination between pH-sensitive and pH-insensitive luciferases could be different.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.

A relationship between Lewis (a-b-) phenotype and the metabolic syndrome X has been suggested. We studied the frequency of Lewis (a-b-) phenotype in subjects with non-insulin-dependent diabetes mellitus (NIDDM) as well as the relationship between Lewis phenotype and lipid concentration in NIDDM patients. Lewis red blood cell phenotyping was done in 207 NIDDM subjects and 345 non-diabetic control subjects by immuno-agglutination with anti-Lewis a and b monoclonal antibodies. Among NIDDM patients, the proportion with the Lewis (a-b-) phenotype was significantly increased (23.6% vs 14.3%, p = 0.01), and this phenotype was associated with higher levels of triglycerides (2.40 +/- 2.58 vs 1.97 +/- 1.25, p = 0.03). This study shows a relationship between NIDDM and Lewis (a-b-) phenotype. Hypertriglyceridaemia in Lewis-negative NIDDM could suggest an increased risk of ischaemic heart disease for these subjects.

Two groups of 11 ICU respiratory patients ventilated with PSV have been sedated with propofol (group I) or with midazolam (group II). After the endovenous administration of the induction dose (propofol 1.5 mg/kg; midazolam 0.15 mg/kg) sedation was obtained with continuous infusion of the drugs (propofol 2 mg/kg/h; midazolam 0.24 mg/kg/h). In this setting the Authors evaluated the level of sedation (Ramsey scale) and the side effect of the two drugs. At induction midazolam caused a reduction of tidal volume for some minutes and a greatest sedation in comparison with propofol, while propofol caused reduction of MAP (p < 0.01) and transitory apnoea. Even if during the infusion of propofol the level of sedation decreased with time (p < 0.05; y = -0.0357 x + 3.07) it was more stable in comparison with that registered during continuous infusion of midazolam (p < 0.01; y = -0.2018 x + 5.19.